4-(4Methoxybenzyl)-n′-(5-nitro1,3-thiazol-2-yl)urea and its use in the treatment of conditions associated with glycogen-synthase kinase-3 (gsk3)

ABSTRACT

The present invention relates to a new compound of formula I as a free base or a pharmaceutically acceptable salt thereof, a process for the preparation, pharmaceutical formulations containing said therapeutically active compound and to the use of said compound in therapy.

FIELD OF THE INVENTION

The present invention relates to a new compound of formula I, as a freebase or a pharmaceutically acceptable salt thereof, to pharmaceuticalformulations containing said compound and to the use of said activecompound in therapy. The present invention further relates to a processfor the preparation of the compound of formula I.

An object of the invention is to provide a compound of formula I fortherapeutic use, especially a compound that is useful for the preventionand/or treatment of conditions associated with glycogen synthasekinase-3 (GSK3) in mammals, including man. Particularly, a compound offormula I exhibiting a selective inhibition of GSK-3.

BACKGROUND OF THE INVENTION

Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinasecomposed of two isoforms (α and β), which are encoded by distinct genesbut are highly homologous within the catalytic domain. GSK3 is highlyexpressed in the central and peripheral nervous system. GSK3phosphorylates several substrates including tau, β-catenin, glycogensynthase, pyruvate dehydrogenase and elongation initiation factor 2b(eIF2b). Insulin and growth factors activate protein kinase B, whichphosphorylates GSK3 on serine 9 residue and inactivates it.

Alzheimer's Disease (AD) Dementias, and Taupathies.

AD is characterized by cognitive decline, cholinergic dysfunction andneuronal death, neurofibrillary tangles and senile plaques consisting ofamyloid-β deposits. The sequence of these events in AD is unclear, butbelieved to be related. Glycogen synthase kinase 3β (GSK3β) or Tau (τ)phosphorylating kinase selectively phosphorylates the microtubuleassociated protein τ in neurons at sites that are hyperphosphorylated inAD brains. Hyperphosphorylated protein τ has lower affinity formicrotubules and accumulates as paired helical filaments, which are themain components that constitute neurofibrillary tangles and neuropilthreads in AD brains. This results in depolymerization of microtubules,which leads to dying back of axons and neuritic dystrophy.Neurofibrillary tangles are consistently found in diseases such as AD,amyotrophic lateral sclerosis, parkinsonism-dementia of Gaum,corticobasal degeneration, dementia pugilistica and head trauma, Down'ssyndrome, postencephalatic parkinsonism, progressive supranuclear palsy,Niemann-Pick's Disease and Pick's Disease. Addition of amyloid-β toprimary hippocampal cultures results in hyperphosphorylation of τ and apaired helical filaments-like state via induction of GSK3β activity,followed by disruption of axonal transport and neuronal death (Imahoriand Uchida., J. Biochem 121:179–188, 1997). GSK3β preferentially labelsneurofibrillary tangles and has been shown to be active in pre-tangleneurons in AD brains. GSK3 protein levels are also increased by 50% inbrain tissue from AD patients. Furthermore, GSK3β phosphorylatespyruvate dehydrogenase, a key enzyme in the glycolytic pathway andprevents the conversion of pyruvate to acetyl-Co-A (Hoshi et al., PNAS93:2719–2723, 1996). Acetyl-Co-A is critical for the synthesis ofacetylcholine, a neurotransmitter with cognitive functions. Thus, GSK3βinhibition may have beneficial effects in progression as well as thecognitive deficits associated with Alzheimer's disease and otherabove-referred to diseases.

Chronic and Acute Neurodegenerative Diseases

Growth factor mediated activation of the PI3K/Akt pathway has been shownto play a key role in neuronal survival. The activation of this pathwayresults in GSK3β inhibition. Recent studies (Bhat et. al., PNAS97:11074–11079 (2000)) indicate that GSK3β activity is increased incellular and animal models of neurodegeneration such as cerebralischemia or after growth factor deprivation. For example, the activesite phosphorylation was increased in neurons vulnerable to apoptosis, atype of cell death commonly thought to occur in chronic and acutedegenerative diseases such as Alzheimer's Disease, Parkinson's Disease,amyotrophic lateral sclerosis, Huntington's Disease and HIV dementia,ischemic stroke and head trauma. Lithium was neuroprotective ininhibiting apoptosis in cells and in the brain at doses that resulted inthe inhibition of GSK3β. Thus GSK3β inhibitors could be useful inattenuating the course of neurodegenerative diseases.

Bipolar Disorders (BD)

Bipolar Disorders are characterised by manic episodes and depressiveepisodes. Lithium has been used to treat BD based on its moodstabilising effects. The disadvantage of lithium is the narrowtherapeutic window and the danger of overdosing that can lead to lithiumintoxication. The recent discovery that lithium inhibits GSK3 attherapeutic concentrations has raised the possibility that this enzymerepresents a key target of lithium's action in the brain (Stambolic etal., Curr. Biol. 6:1664–1668, 1996; Klein and Melton; PNAS 93:8455–8459,1996). Inhibition of GSK3β may therefore be of therapeutic relevance inthe treatment of BD as well as in AD patients that have affectivedisorders.

Schizophrenia

GSK3 is involved in signal transduction cascades of multiple cellularprocesses, particularly during neural development. Kozlovsky et al (Am JPsychiatry 2000 May; 157(5):831–3) found that GSK3β levels were 41%lower in the schizophrenic patients than in comparison subjects. Thisstudy indicates that schizophrenia involves neurodevelopmental pathologyand that abnormal GSK3 regulation could play a role in schizophrenia.Furthermore, reduced β-catenin levels have been reported in patientsexhibiting schizophrenia (Cotter et al., Neuroreport 9:1379–1383(1998)).

Diabetes

Insulin stimulates glycogen synthesis in skeletal muscles via thedephosphorylation and thus activation of glycogen synthase. Underresting conditions, GSK3 phosphorylates and inactivates glycogensynthase via dephosphorylation. GSK3 is also over-expressed in musclesfrom Type II diabetic patients (Nikoulina et al., Diabetes 2000February; 49(2):263–71). Inhibition of GSK3 increases the activity ofglycogen synthase thereby decreasing glucose levels by its conversion toglycogen. GSK3 inhibition may therefore be of therapeutic relevance inthe treatment of Type I and Type II diabetes and diabetic neuropathy.

Hair Loss

GSK3 phosphorylates and degrades β-catenin. β-catenin is an effector ofthe pathway for keratonin synthesis. β-catenin stabilisation may be leadto increase hair development. Mice expressing a stabilised β-catenin bymutation of sites phosphorylated by GSK3 undergo a process resembling denovo hair morphogenesis (Gat et al., Cell Nov. 25, 1998; 95(5):605–14)). The new follicles formed sebaceous glands and dermalpapilla, normally established only in embryogenesis. Thus GSK3inhibition may offer treatment for baldness.

Oral Contraceptives

Vijajaraghavan et al. (Biol Reprod 2000 June; 62 (6):1647–54) reportedthat GSK3 is high in motile versus immotile sperm. Immunocytochemistryrevealed that GSK3 is present in the flagellum and the anterior portionof the sperm head. These data suggest that GSK3 could be a key elementunderlying motility initiation in the epididymis and regulation ofmature sperm function. Inhibitors of GSK3 could be useful ascontraceptives for males.

DISCLOSURE OF THE INVENTION

The object of the present invention is to, provide a compound having aselective inhibiting effect at GSK3 as well as having a goodbioavailability.

Accordingly, the present invention provides a compound of formula I:

as a free base or a pharmaceutically acceptable salt thereof.

A suitable pharmaceutically acceptable salt of the compound of theinvention is, for example, an acid-addition salt, which is sufficientlybasic, such as an inorganic or organic acid or an alkali metal salt, analkaline earth metal salt.

The compound of formula I may be administered in the form of a pro-drug,which is broken down in the human or animal body to give the compound offormula I.

It is also to be understood that the compound of formula I can exist inthe solvated form such as the hydrated form, as well as in theunsolvated form. It is to be understood that the invention encompassesall such forms.

Methods of Preparation

Another aspect of the present invention provides a process for preparingthe compound of formula I as a free base or a pharmaceuticallyacceptable salt thereof.

The process for the preparation of the compound of formula I comprisesof:

reacting a compound of formula II with a compound of formula III in asuitable solvent such as N,N-dimethylformamide, dimethyl sulfoxide,dioxane or tetrahydrofuran at a temperature within the range of +70 to+150° C.

EXAMPLE 1 N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea

A mixture of 2-amino-5-nitrothiazole (0.89 g, 6.13 mmol) and4-methoxybenzylisocyanate (1 g, 6.13 mmol) in N,N-dimethylformamide (6mL) was heated at 100° C. under nitrogen atmosphere for 15 h. Themixture was allowed to cool and was partitioned between water and ethylacetate. The aqueous layer was extracted with another portion of ethylacetate. The combined organic layers were washed with brine, dried(MgSO₄) and evaporated to give 2.5 g of a semi-solid crude product. Mostof the material was dissolved in chloroform/ethanol (98:2, approx. 15mL) and triethylamine (3 mL) followed by filtration. The dissolved crudeproduct was purified on a silica gel column using CHCl₃/Etanol, 95:5 asthe eluent to give 408 mg (22% yield) of the title compound as ayellowish solid: mp >190° C. (decomp.); ¹H NMR (DMSO-d₆, 400 MHz) δ11.64 (br s, 1 H), 8.50 (s, 1 H), 7.25–7.23 (m, 3 H), 6.92–6.89 (m, 2H), 4.30 (d, J=5.9 Hz, 2 H), 3.73 (s, 3 H); ¹³CNMR (DMSO-d₆, 100 MHz) δ164.42, 158.43, 153.48, 143.47, 140.80, 130.82, 128.72, 113.82, 55.08,42.60; ESMS m/z 309 (M⁺+1).

Pharmaceutical Formulations

According to one aspect of the present invention there is provided apharmaceutical formulation comprising the compound of formula I, as afree base or a pharmaceutically acceptable salt thereof, for use in theprevention and/or treatment of conditions associated with glycogensynthase kinase-3.

The formulation may be in a form suitable for oral administration, forexample as a tablet, pill, syrup, powder, granule or capsule, forparenteral injection (including intravenous, subcutaneous,intramuscular, intravascular or infusion) as a sterile solution,suspension or emulsion, for topical administration as an ointment, patchor cream or for rectal administration as a suppository.

In general the above formulations may be prepared in a conventionalmanner using pharmaceutically acceptable carriers and diluents.

Suitable daily doses of the compound of formula I in the treatment of amammal, including man are approximately 0.01 to 250 mg/kg bodyweight atperoral administration and about 0.001 to 250 mg/kg bodyweight atparenteral administration. The typical daily dose of the activeingredient varies within a wide range and will depend on various factorssuch as the relevant indication, the route of administration, the age,weight and sex of the patient and may be determined by a physician.

Medical Use

Surprisingly, it has been found that the compound of the presentinvention, as a free base or a pharmaceutically acceptable salt thereof,is well suited for inhibiting glycogen synthase kinase-3 (GSK3).Accordingly, the compound of the present invention is expected to beuseful in the prevention and/or treatment of conditions associated withglycogen synthase kinase-3 activity, i.e. the compound may be used toproduce an inhibitory effect of GSK3 in mammals, including man, in needof such prevention and/or treatment.

GSK3 is highly expressed in the central and peripheral nervous systemand in other tissues. Thus, it is expected that the compound of theinvention is well suited for the prevention and/or treatment ofconditions associated with glycogen synthase kinase-3 in the central andperipheral nervous system. In particular, the compound of the inventionis expected to be suitable for prevention and/or treatment of one ormore conditions such as dementia, Alzheimer's Disease, Parkinson'sDisease, Frontotemporal dementia Parkinson's Type, Parkinson dementiacomplex of Gaum, HIV dementia, diseases with associated neurofibrillartangle pathologies, amyotrophic lateral sclerosis, corticobasaldegeneration, dementia pugilistica, Down's syndrome, Huntington'sDisease, postencephelatic parkinsonism, progressive supranuclear palsy,Pick's Disease; Niemann-Pick's Disease, stroke, head trauma and otherchronic neurodegenerative diseases, Bipolar Disease, affectivedisorders, depression, schizophrenia, cognitive disorders, Type I andType II diabetes and diabetic neuropathy, hair loss and contraceptivemedication.

The dose required for the therapeutic or preventive treatment of aparticular disease will necessarily be varied depending on the hosttreated, the route of administration and the severity of the illnessbeing treated.

The present invention relates also to the use of the compound of formulaI, in the manufacture of a medicament for the prevention and/ortreatment of conditions associated with GSK3.

In the context of the present specification, the term “therapy” includestreatment as well as prevention, unless there are specific indicationsto the contrary. The terms “therapeutic” and “therapeutically” should beconstrued accordingly.

The invention also provides a method of treatment and/or prevention ofconditions associated with GSK3, in a patient suffering from, or at riskof said condition, which comprises administering to the patient aneffective amount of the compound of formula I.

Non-Medical Use

In addition to their use in therapy, the compound of formula I, as afree base or a pharmaceutically acceptable salt thereof, is also usefulas a pharmacological tool in the development and standardisation of invitro and in vivo test systems for the evaluation of the effects ofinhibitors of GSK3 in laboratory animals such as cats, dogs, rabbits,monkeys, rats and mice, as part of the search for new therapeuticalagents.

Pharmacology

Determination of ATP Competition in Scintillation Proximity GSK3β Assay.

GSK3βScintillation Proximity Assay.

The competition experiments were carried out in duplicate with 10different concentrations of the inhibitors in clear-bottom microtiterplates (Wallac, Finland). A biotinylated peptide substrate,Biotin-Ala-Ala-Glu-Glu-Leu-Asp-Ser-Arg-Ala-Gly-Ser(PO₃H₂)-Pro-Gln-Leu(SEQ ID NO: 1) (AstraZeneca, Lund), was added at a final concentrationof 1 μM in an assay buffer containing 1 mU recombinant human GSK3β(Dundee University, UK), 12 mM morpholinepropanesulfonic acid (MOPS), pH7.0, 0.3 mM EDTA, 0.01% β-mercaptorethanol, 0.004 % Brij 35 (aphosphatase inhibitor), 0.5% glycerol and 0.5 μg BSA/25 μl. The reactionwas initiated by the addition of 0.04 μCi [γ-³³P]ATP (Amersham, UK) andunlabelled ATP at a final concentration of 1 μM and assay volume of 25μl. After incubation for 20 minutes at room temperature, each reactionwas terminated by the addition of 25 μl stop solution containing 5 mMEDTA, 50 μM ATP, 0.1 % Triton X-100 and 0.25 mg streptavidin coatedScintillation Proximity Assay (SPA) beads (Amersham, UK). After 6 hoursthe radioactivity was determined in a liquid scintillation counter (1450MicroBeta Trilux, Wallac). The inhibition curves were analysed bynon-linear regression using GraphPad Prism, USA. The K_(m) value of ATPfor GSK3β, used to calculate the inhibition constants (K_(i)) of thevarious compounds, was 20 μM.

The following abbreviations have been used:

MOPS Morpholinepropanesulfonic acid EDTA Ethylenediaminetetraacetic acidBSA Bovin Serum Albumin ATP Adenosine Triphophatase SPA ScintillationProximity Assay GSK3 Glycogen synthase kinase 3Results

The K_(i) value for the compound of the present invention is 96 nM.

1. A compound of formula I

in the form of a free base or a pharmaceutically acceptable saltthereof.
 2. A pharmaceutical formulation comprising as active ingredienta therapeutically effective amount of the compound of claim 1 inassociation with pharmaceutically acceptable carriers or diluents.
 3. Amethod for the inhibition or treatment of a medical condition associatedwith glycogen synthase kinase-3, the method comprising administering toa patient in need of such inhibition or treatment a therapeuticallyeffective amount of the compound or salt thereof as defined in claim 1,wherein the condition is dementia or Alzheimer's Disease.
 4. A processfor the preparation of the compound of formula I, the process comprisingreacting the compound of formula II with the compound of formula III.


5. The method according to claim 3, wherein the compound is administeredperorally.
 6. The method according to claim 5, wherein thetherapeutically effective daily dosage is about 0.01 to about 250 mg/kg.7. The method according to claim 3, wherein the compound is administeredparenterally.
 8. The method according to claim 7, wherein thetherapeutically effective daily dosage is about 0.001 to about 250mg/kg.
 9. The process according to claim 4, wherein a compound offormula II is reacted with a compound of formula III in a solvent at atemperature from about +70° C. to about +150° C.
 10. The processaccording to claim 4 or 9, wherein the solvent is N,N-dimethylformamide,dimethyl sulfoxide, dioxane or tetrahydrofuran.